Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the equilibration of carbon dioxide, protons, and bicarbonate. For several acid/base-coupled membrane carriers it has been shown that the catalytic activity of CA supports transport activity, an interaction coined "transport metabolon." We have reported that CA isoform II (CAII) enhances lactate transport activity of the monocarboxylate transporter isoform I (MCT1) expressed in Xenopus oocytes, which does not require CAII catalytic activity (Becker, H. M., Fecher-Trost, C., Hirnet, D., SĂŒlt-emeyer, D., and Deitmer, J. W. (2005) J. Biol. Chem. 280, 39882-39889). Coexpression of MCT1 with either wild type CAII or the catalytically inactive mutant CAII-V143Y similarly enhanced MCT1 activity, although injection of CAI or coexpression of an N-terminal mutant of CAII had no effect on MCT1 transport activity, demonstrating a specific, nonenzymatic action of CAII on lactate transport via MCT1. If the H Ű gradient was set to dominate the rate of lactate transport by applying low concentrations of lactate at a high H Ű concentration, the effect of CAII was largest. We tested the hypothesis of whether CAII helps to shuttle H Ű along the inner face of the cell membrane by measuring the pH change with fluorescent dye in different areas of interest during focal lactate application. Intracellular pH shifts decayed from the focus of lactate application to more distant sites much less when CAII had been injected. We present a hypothetical model in which the effective movement of H Ű into the bulk cytosol is increased by CAII, thus slowing the dissipation of the H Ű gradient across the cell membrane, which drives MCT1 activity.Transport of acid/base equivalents across cell membranes plays a crucial role both for pH regulation and cellular import and export of metabolites. Many Na Ï© -dependent and Na Ï© -independent transport modes of metabolites (e.g. neurotransmitter substances) also transport H Ï© , OH ÏȘ , or HCO 3 ÏȘ as co-or counter-substrate. The energetic compounds lactate and pyruvate are primarily transported by monocarboxylate transporters (MCT) 2 in an electroneutral 1 proton-1 organic anion transport mode. Members of the MCT family, which belong to the human gene family SLC16 which comprise 14 isoforms, are expressed in most tissues, in particular those with a large energy consumption like muscle and brain (1, 2). In the brain MCT1 is located mainly in glial cells, where it facilitates the export of lactate, which is then taken up by neurons via the high affinity MCT2; thereby the two isoforms are believed to shuttle lactate from glial cells to neurons which seems to be pivotal for brain energy metabolism (3-6). In muscle, MCT1 is highly expressed in oxidative type I fibers, where it mediates import of lactate that is released by glycolytic muscle cells via MCT3 and MCT4 (7-9). MCT1, when expressed in Xenopus oocytes, operates with a K m value of 3.5 mM for lactate influx and of 6.4 mM for efflux of lactate. Both the rate and the amplitude of the lactate-induced ac...